Moringa oleifera is a tropical plant with high nutritional and medicinal value. Recent studies have reported its remarkable effects in inflammatory, antioxidative, and anti‐diabetes modulations, but there was no significant report on its role in animal breeding. In this study, we investigated the effects of dietary Moringa oleifera leaf (MOL) on reproductive performances in mice. We studied the reproductive performance of mice for six consecutive gestations. Mice fed with 4% MOL diet showed improved litter size, litter birth weight, and litter survivals until weaning age compared to control mice fed with normal diet (p < 0.05). Mice fed with MOL diet did not change weight and organ coefficients. Serum malondialdehyde (MDA) concentrations in both male and female mice were significantly decreased by dietary MOL (p < 0.05), but glutathione peroxidase (GSH‐PX) and superoxide dismutase (SOD) were unchanged. For male, dietary MOL significantly reduced sperm abnormality rate (p < 0.05) and Bcl2‐associated X protein (Bax) expression in testis (p < 0.05), but did not affect serum testosterone and the expression levels of androgen receptor (AR), phosphoglycerate kinase 2 (Pgk2), protamine2 (Prm2), and B cell leukemia/lymphoma 2 (Bcl2) in testis. For female, dietary MOL did not change serum estradiol and the expressions of estrogen receptor beta (ERβ), Bcl2, Bax, and vascular endothelial growth factor receptor (VEGFR) in ovary. In summary, MOL increased litter size and antioxidant ability, reduced the rate of sperm abnormality and the expression of Bax. Therefore, MOL may serve as a functional feed addictive for improving animal reproductive performance.

Secretory immunoglobulin A (SIgA) plays an important role in gut acquired immunity and mucosal homeostasis. Breast milk is the irreplaceable nutritional source for mammals after birth. Current studies have shown the potential functional role of milk-derived small extracellular vesicles (sEVs) and their RNAs cargo in intestinal health and immune regulation. However, there is a lack of studies to demonstrate how milk-derived sEVs affect intestinal immunity in recipient. In this study, through in vivo experiments, we found that porcine milk small extracellular vesicles (PM-sEVs) promoted intestinal SIgA levels, and increased the expression levels of polymeric immunoglobulin receptor (pIgR) both in mice and piglet. We examined the mechanism of how PM-sEVs increased the expression level of pIgR in vitro by using a porcine small intestine epithelial cell line (IPEC-J2). Through bioinformatics analysis, dual-luciferase reporter assays, and overexpression or knockdown of the corresponding non-coding RNAs, we identified circ-XPO4 in PM-sEVs as a crucial circRNA, which leads to the expression of pIgR via the suppression of miR-221-5p in intestinal cells. Importantly, we also observed that oral administration of PM-sEVs increased the level of circ-XPO4 and decreased the level of miR-221-5p in small intestine of piglets, indicating that circRNAs in milk-derived sEVs act as sponge for miRNAs in recipients. This study, for the first time, reveals that PM-sEVs have a capacity to stimulate intestinal SIgA production by delivering circRNAs to receptors and sponging the recipient’s original miRNAs, and also provides valuable data for insight into the role and mechanism of animal milk sEVs in intestinal immunity.

Dietary protein sources have profound effects on children and young animals, and are important for the gut barrier function and immune resilience. Milk and soy are the main sources of protein for children and young animals after weaning. The objective of this study was to compare the effects of dairy and soy proteins on the intestinal barrier in early development. Weanling C57BL/6 mice were fed AIN-93G diets prepared with casein or soy protein isolate (SPI) for 21 days. Compared with those fed with the casein diet, mice fed with the SPI diet did not change their body weight and organ coefficients, but increased their feed intake and ratio of feed to gain. SPI lowered the level of luminal secretory immunoglobulin A (SIgA) and downregulated the levels of IL-4, IL-13, polymeric immunoglobulin receptor (Pigr), Janus kinase 1 (Jak1), signal transducer and activator of transcription 6 (Stat6), and transforming growth factor-β (Tgfb) in the mouse ileum. Western blotting of ileal proteins confirmed that SPI suppressed the activation of the JAK1/STAT6 signaling pathway. Furthermore, SPI attenuated intestinal mucin production, as demonstrated by the decreased numbers of intestinal goblet cells and the reduced relative expression levels of mucin 1 (Muc1), mucin 2 (Muc2), trefoil factor 3 (Tff3), glucose-regulated protein 94 (Grp94), and anterior gradient homolog 2 (Agr2). The results indicated that the SPI diet could attenuate mouse intestinal immunity, as demonstrated by decreased SIgA and mucin production in the intestine. Therefore, we suggest that our findings should be of consideration when SPI or casein are used as dietary protein sources.

Skeletal muscle development at the embryonic stage is critical for muscle growth in poultry, during which microRNAs (miRNAs) play a vital role. Geese are one of the main poultry species, but how miRNAs regulate skeletal muscle development in geese during the embryonic stage remains poorly understood. This study characterized histological features and miRNA expression profiles during leg muscle development in Daozhou Grey goose (a prized Chinese breed) embryos at three stages (E14, E21, and E28). The objective was to identify differentially expressed miRNAs (DEMs) associated with muscle development in Daozhou Grey goose and utilize bioinformatics to predict the potential biological functions of these DEMs. Histological observations revealed stage-specific muscle development, marked by myoblast proliferation at E14, differentiation at E21 and muscle fiber maturation at E28. A total of 340 known and 270 novel miRNAs were identified by miRNA sequencing, with miR-148a-3p, miR-1a-3p, miR-100-5p, miR-206, and miR-92-3p being the most abundant. Furthermore, differential expression analysis identified 105, 107 and 70 DEMs across E14 vs E21, E14 vs E28, and E21 vs E28 comparisons, respectively. The expression patterns of the twelve randomly selected DEMs (9 known and 3 novel miRNAs) were validated by Quantitative real-time polymerase chain reaction (qRT-PCR), confirming the sequencing results. Functional enrichment analysis of predicted miRNA target genes revealed involvement of the MAPK, TGF-β and Notch signaling pathways in embryonic myogenesis. The miRNA-mRNA interaction network analysis of the top 10 most DEMs and their target genes revealed key regulatory nodes such as let-7k-5p-MAP3K1, miR-133a-3p-FZD7, miR-133c-3p-STAT3, miR-187-3p-ZEB2, and miR-205b-SETD3, with inverse expression patterns validated by qRT-PCR. It was hypothesized that the above pathways as well as these miRNA-mRNA target relationships play an important role in the regulating embryonic muscle growth and development of the Daozhou Grey goose. These findings provide the first comprehensive miRNAs profile of embryonic leg muscle in vivo in geese, offering a basic reference and theoretical framework for further research on molecular regulatory mechanisms of goose muscle development.